BioSci 203 lecture 28 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203...

BioSci 203 lecture 28 page 1 ©copyright Bruce Blumberg 2001. All rights reserved

Bio Sci 203 Lecture 28 - cDNA library screening

• Bruce Blumberg ([email protected])– office – 2113E McGaugh Hall– 824-8573– lab (x46873,x43116)– office hours Wednesday 11-12.

• http://blumberg-serv.bio.uci.edu/bio203-w2002/index.htm• http://blumberg.bio.uci.edu/bio203-w2002/index.htm

• Link is also main class web site

• Today– wrap up cDNA library screening– characterization of clones obtained from screening– Protein protein binding assays

http://blumberg-serv.bio.uci.edu/bio203-w2002/index.htm










http://blumberg.bio.uci.edu/bio203-w2002/index.htm









mRNA frequency and cloning

• mRNA frequency classes – classic references

• Bishop et al., 1974 Nature 250, 199-204• Davidson and Britten, 1979 Science 204, 1052-1059

– abundant • 10-15 mRNAs that together represent 10-20% of the total RNA

mass• > 0.2%

– intermediate • 1,000-2,000 mRNAs together comprising 40-45% of the total• 0.05-0.2% abundance

– rare • 15,000-20,000 mRNAs comprising 40-45% of the total• abundance of each is less than 0.05% of the total• some of these might only occur at a few copies per cell

• How does one go about identifying genes that might only occur at a few copies per cell?


Normalization and subtraction• How to identify genes that might only occur at a few copies per cell?

– Improve your chances - alter the representation of the cDNAs

• Normalization - reducing the frequency of abundant and increasing the frequency of rare mRNAs - Bonaldo et al., 1996 Genome Research 6, 791-806– Theoretically, normalization brings all cDNAs into the same order of

magnitude abundance, i.e., within 10 fold of each other• rarely works this well. • Typically, abundant genes reduced 10x, rare ones increased 3-10x• Intermediate class genes do not change much at all

– Approach• make a population of cDNAs single stranded

– tester

• hybridize with a large excess of cDNA or mRNA to Cot =5.5

– driver

• Cot value is critical for success of normalization

– 5-10 is optimal– higher values are not better


Normalization and subtraction (contd)

– Approach (contd)• various approaches to make driver

– use mRNA - may not be easy to get– make ssRNA by transcribing library– ssDNA from gene II/ExoIII treating inserts from plasmid

library– PCR amplification of library

• best approach is to use driver derived from the same library by PCR

– rapid, simple and effective– other approaches each have various technical difficulties– see the Bonaldo review for details.



– What are normalized libraries good for?• EST sequencing• gene identification

– biggest use is to reduce the number of cDNAs that must be screened

– good general purpose target to screen» subtracted libraries are useful but limited in utility

– Drawbacks• Not trivial to make• Size distribution of library changes

– Longer cDNAs lost


Normalization and subtraction (contd)• Subtraction - removing cDNAs (mRNAs) expressed in two populations

leaving only differentially expressed– Sagerström et al. (1997) Ann Rev. Biochem 66, 751-783

• +/- screening St. John and Davis (1979) Cell 16, 443-452. – Hybridize the same library with probes prepared from two

different sources and compare the results• example - hybridize normal liver cDNA library with probes

from normal and cancerous liver

– Colonies or plaques that are expressed in target tissue (tumor) compared with control are picked

– Why aren’t all colonies labeled in normal tissue?


Normalization and subtraction (contd)• What about rare mRNAs? These might be differential but not

abundant enough to detect– Do reverse experiment -> select for absence of a signal– example - hybridize a tumor cDNA library with probe

prepared from normal liver

– select for genes absent in tumor» Get genes lost from normal tissue and gained in

tumor by this approach



• +/- screening (contd)– Advantages

• Relatively simple approach• Doesn’t require difficult manipulations on probes

– Disadvantages• Housekeeping genes often appear to be differential• Sensitivity less than subtracted screening

– +/- screening typically requires >10 fold difference in expression levels using standard methods

• not widely used any longer BUT– microarray analysis is really just a refined version of +/-

screening• fluorescence ratios give good internal standards

– more precise quantitation– increased sensitivity


DNA microarray


Normalization and subtraction (contd)• Subtractive screening - Sargent and Dawid (1983) Science 222, 135-

139. – Make 1st strand cDNA from a tissue and then hybridize it to excess

mRNA from another• larger Cot is best >20 at least

– remove double stranded materials -> common seqs– make a probe or library from the remaining single stranded cDNA– 10-100 fold more sensitive than +/- screening



• Subtractive screening (contd)– benefits

• sensitive• can simultaneously identify all cDNAs that are differentially

present in a population• good choice for identifying unknown, tissue specific genes

– drawbacks• easy to have abundant housekeeping genes slip through

– multistage subtraction is best– in effect normalize first, then subtract

• libraries have limited applications– may not be useful for multiple purposes



– rule of thumb• make a high quality representative library from a tissue of

interest• save subtraction and other fancy manipulations for making

probes to screen such libraries with– unlimited screening– easy to use libraries for different purposes, e.g. the liver

library» hepatocarcinoma» cirrhosis» regeneration specific genes


How to identify your gene of interest• Screening methods depend on what type of information you have in

hand.– Related gene from another species?

– A piece of genomic DNA?

– A mutant

– A functional assay?

– An antibody?

– A partial amino acid sequence?

– A DNA element required for expression of an interesting gene?

– An interacting protein?

– A specific tissue or embryonic stage?

• Low stringency hybridization

• Hybridization

• Complementation• Positional cloning

• Expression screening

• Expression library screening

• Oligonucleotide screening

• Various binding protein strategies

• Interaction screening

• Subtracted or +/- screening


How to identify your gene of interest (contd)• What is the most important piece of information you need to clone a

cDNA?

• First step in any hybridization based method (high or low stringency) is to get information on expression– high stringency homologous screening - Northern analysis– cross species screening requires more care

• perform a genomic Southern to identify hybridization and washing conditions that identify a small number of hybridizing fragments

– standard conditions - 1 M Na+, 43% formamide, 37° C– begin washing at RT in 2 x SSC and expose– increase stringency until signal/noise ratio is acceptable– use these conditions for Northern.

• If Northern is unsuccessful - obtain a genomic clone and repeat the screening at high stringency

– this approach will never fail to identify a homologous gene

– Information on where the mRNA is expressed• either what tissue or• what time during development

– such information is indispensable!!


How to identify your gene of interest (contd)• Cloning by complementation

– generally only useful with manipulable genetic systems• yeast• Drosophila• C. elegans• zebrafish

– presumes that complemented mutant is readily observable– Approach

• transfer pooled cDNA libraries in expression vectors into the mutant

– or mRNA pools derived from libraries• assay for rescue• subdivide positive pools and repeat

– advantages• direct functional test • rapid compared with chromosome walking

– disadvantages• fairly tedious• dependent on library quality• requires easily observable rescue


How to identify your gene of interest (contd)• Positional cloning

– If your mutant results from a transposon insertion then this can be recovered

– If insertion is a P-element or such• Make genomic library from mutant

– What type of library will you make? Why?• Screen with transposon

– Recover positives, sequence flanking region• Use flanking sequence to screen normal genomic library

– What type of library will you screen?– If insertion is a gene trap or related

• You can digest mutant DNA with an enzyme that linearizes the vector

• Ligate and transform• Colonies that form should have flanking region

– sequence• Use this to screen normal library• OR• Use inverse PCR to get flanking sequence from plasmid and

use this to probe library


How to identify your gene of interest (contd)• Functional screening (expression cloning)

– if you have a functional assay, expression cloning is reasonable– strategy:

• Large pools (~10,000) of cDNAs tested for function– microinjection– transfection– receptor binding (panning)

• positive pools are subdivided and retested to obtain pure cDNAs

• cycle is repeated until single clones obtained– Advantages

• functional approach• in vivo testing is possible• can identify secreted proteins and receptors

– Disadvantages• Slow and tedious• sensitivity issue due to pool size• extensive retesting of pools is required

– applications:• many receptors and transporters cloned this way


How to identify your gene of interest (contd)• Antibody screening of cDNA expression libraries

– requirements• antibody must recognize denatured epitope (western blot)

– many monoclonals recognize 3-D or sugar epitopes• affinity purified antibodies work best• cDNA expression library, e.g., λgt11 series

– approach• plate library and induce replicate filters• incubate with antibody, wash and develop the filters• repeat until a pure clone is obtained

– verification• affinity purify antibody with phage fusion protein – western

with original protein– advantages

• best choice if only antibody is available– disadvantages

• λgt11 and relatives are painful to work with• your antibody may not be suitable

– sugar directed– structural epitope


How to identify your gene of interest (contd)

• A partial amino acid sequence?– Purified protein and have one or more partial amino acid

sequences• make a peptide antibody and screen (slow)• Oligonucleotide screening based on aa sequence

– multiple codons for most aa• PCR between multiple primers

– three types of oligos in use• long guess-mers - pick the wobble base

– relies on low stringency hybridization• inosine - use inosine for multiple bases

– I:C >> others• degenerate oligos (mixtures of all possible seqs)

– mixtures of < 1024 virtually always work


How to identify your gene of interest (contd)• A partial amino acid sequence (contd)?

– approach• pick an aa sequence that predicts a reasonable probe

complexity (~1024 fold)(avoid ser, leu, arg) WHY?• synthesize fully degenerate mixture of probes and label• hybridize at low stringency (Tm-25 for the most AT rich

sequence)• wash at high stringency in 3M tetramethylammonium chloride

– TMAC stabilizes AT base pairs -> melting temperature is a strict function of length

– works best for 21-23 mers– degenerate oligo and TMAC

• advantages– degenerate oligos always work– fast– only requires a single sequence

• disadvantages– TMAC method requires strict adherence to technique– aa sequence may not predict a good oligo

» e.g., too many leu, ser or arg



• A partial amino acid sequence (contd)– PCR – design primers to two conserved sequences, amplify, clone

• advantages – very fast– almost anyone can manage

• disadvantages– requires 2 good sequences– PCR errors may give incorrect sequence



• A DNA element required for expression of an interesting gene?– How to identify what factors bind to putative elements?

• examine the sequence– does it contain known binding sites?– Check TRANSFAC database– http://www.gene-regulation.com/pub/databases.html#tran

sfac» if yes, do such proteins bind to the isolated element in

gel-shift experiments?• do the elements bind proteins from nuclear extracts?

– gel shift (EMSA) experiments• clone the elements into reporters with minimal promoters.

– do these constructs recapitulate activity?– What does the sequence tell you about the binding protein?

• AGGTCATGACCT–

http://www.gene-regulation.com/pub/databases.html#transfac

http://www.gene-regulation.com/pub/databases.html#transfac



• Biochemical purification of binding proteins– tedious, considerable biochemical skill required– two basic approaches

• fractionate nuclear extracts chromatographically and test fractions for ability to bind the element

• DNA-affinity chromatography– multimerize the element and bind to a resin– pass nuclear extracts across column and purify specific

binding proteins– protein microsequencing– predict DNA sequence from amino acid sequence

• look in the database• prepare oligonucleotides and screen library



• Biochemical purification of binding proteins (contd)– advantages

• gold standard• if you can purify proteins, this will always work

– not so many good protein biochemists• works for dimeric proteins and complexes

– disadvantages• slow, tedious• need good protein sequencing facility• biochemical expertise required• expense of preparing preparative quantities of nuclear

extracts



• Molecular biological approaches to identifying binding proteins– oligonucleotide screening of expression libraries (Singh

screening)• multimerize oligonucleotide and label with 32P• screen expression library to identify binding proteins• advantages

– straightforward– much less biochemical expertise required than

biochemical purification– relatively fast

• disadvantages– can’t detect binding if multiple partners are required– fair amount of “touch” required

BioSci 203 lecture 28 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203...

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Transcript of BioSci 203 lecture 28 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203...